Autoimmune diabetes is likely preceded by an extended period of silent islet beta-cell destruction. Autoantibody assays aid in the diagnosis and prediction of type 1 diabetes but do not enable confirmation or assessment of ongoing islet cell damage. Metabolic tests reveal only extensive islet damage or immunological rejection of transplanted islet tissue after the fact. In short, there remains a critical need for biomarkers of ongoing islet injury and inflammation (insulitis). By way of contrast, damage to other organs, both prior to and after the onset of clinical symptoms, can be detected and monitored by serum assays for discharged proteins. Autoantibodies against the enzyme GAD65 often precede, sometimes by many years, the onset of autoimmune diabetes. GAD65 is released from cultured, stressed islets. The central hypothesis of this application is that a plasma assay for the beta-cell protein GAD65 will provide, for the first time, a means to detect and monitor ongoing beta-cell damage such as occurs in individuals with autoimmune diabetes, in individuals with asymptomatic insulitis, and in recipients of islet transplants. In the course of preliminary work to test this hypothesis, we have developed what is, to the best of our knowledge, the only biomarker assay capable of detecting ongoing islet damage in vivo. The objective of the project proposed here is to characterize the newly-found role of GAD65 as a circulating marker of islet injury and determine whether discharged GAD65 can serve as a biomarker of insulitis and autoimmunity in type 1 diabetes. The specific aims are: 1. To determine circulating GAD65 levels prior to and at the onset of diabetes in rats treated with high dose and multiple low-doses of streptozotocin and in diabetes-prone BB rats; and 2. To test the hypothesis that subjects with type 1 diabetes have increased plasma GAD65 levels relative to controls at the time of or prior to disease onset. Data generated by these studies will form the basis of further studies of the time-course, triggers and clinical utility of monitoring GAD65 discharge. Our long term objective is identification of circulating biomarkers of islet damage that will become important tools for diabetes-related research and clinical care. Relevance to Public Health: A serum assay capable of detecting diabetes-associated beta cell damage would be invaluable for a variety of applications, including helping identify candidates for preventative therapies, monitoring the response to investigational therapies, assessing drugs for islet toxicity, monitoring transplanted islets for rejection, and investigating the triggers and pathogenesis of islet damage and autoimmunity both in humans and in animal models. [unreadable] [unreadable] [unreadable]